Experimental Study On The Molecular Imaging Of Epidermal Growth Factor Receptor With PET/CT

Epidermal growth factor receptor (EGFR) is over expressed in 90% percent of tumors, such as breast cancer, glioma, laryngeal cancer, carcinoma of the head and neck, and prostate cancer. Signal transduction pathways turned on by EGFR tyrosine kinase (TK) contribute to tumor processes such as cellular proliferation, decreased apoptosis, angiogenesis, and invasion and metastasis. EGFR belongs to a family of proteins involved in the proliferation of normal and malignant cells. Alteration in the expression and activity of growth factor receptors can not only directly perturb growth regulation but may also affect the sensitivity of cancer cells to various cytotoxic treatments, including radioation therapy. Increasing evidence indicates that the EGFR-TK, through activating its downstream signal transduction pathways, EGFR have shown a positive correlation between the levels of EGFR expression and the resistance of tumor to radiation therapy.Thus, the EGFR is an attractive target for the design and development of compounds that can specifically bind the receptor, and inhibit its TK activity and its signal transduction pathway in cancer cells. Such compounds may serve as therapeutic or diagnostic agents. In recent years, with the development of molecular target therapy, EGFR has been the attractive target for the tumor therapy which plays a directory role for tumor therapy, estimate of response and prognosis. So monitoring EGFR expression play a very important role for tumor therapy. EGFR expression has been measured by a variety of techniques, of which the most widely applied is immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), which tumor specimens were derived postoperatively or biopsy accessible. Over the past decades, several in vitro/ex vivo assays have been proposed for the purpose of predicting response to radiotherapy measuring variables such as the surviving fraction 2 Gy (SF2), potential tumor doubling time (Tpot), thymidine labeling index, or tumor hypoxia. These techniques mostly require invasive biopsy also and are invariably subject to sampling errors. Moreover, due to the invasiveness of the procedure, repeated determinations during or after treatment are, in practice, rarely feasible. Consequently, noninvasive alternatives, with repetitive whole body assessment capacity allowing visualization of the whole tumor, would be highly welcomed. Therefore, there is a growing need in oncology for more specific imaging techniques for monitoring EGFR-targeted therapy.With the development and crossing each other of nuclear medicine, molecular biology, imageology. The tracer labled of radionuclide plays a more and more important role in tumor diagnosis, and molecular imaging has been an independent subject of science. Receptor imaging is the most important field of the molecular imaging. Receptor imaging shows the founction and distribution of receptor with the instrument by radioisotope labeling ligand specific binding with the receptor. Functional imaging by means of positron emission tomography (PET) or single photon emission computerized tomography (SPECT) facilitates the evaluation of tumor physiology, metabolism, and proliferation, parameters determining outcome to radiotherapy treatment. PET can be used to image and quantify the in vivo distribution of positron-emitting radioisotopes such as ¹⁵O, ¹¹C, and ¹⁸F that can be substituted or added into biologically relevant compounds. SPECT may be used to image compounds labeled with, e.g., ¹¹¹In, ⁹⁹mTc, and ¹²³I in vivo. PET has a number of advantages over SPECT. Increased sensitivity and better time resolution make dynamic data acquisition easier and better with PET.PET, a nuclear medicine imaging technology, which allows the three-dimensional, quantitative determination of the distribution of positron-emitting radioisotopes within the human body, is becoming an increasingly important tool for the measurement of physiological, biochemical, and pharmacological function at the molecular level or gene level, both in healthy and pathological states. Following injection of a suitable biomarker, the PET scan provides a mapping of the biomarker distribution and hence of a specific receptor, transporter or enzyme in the human body. For example, ¹⁸FDG as a PET tracer showing the level of glycometabolism has been used in clinic widely. In spite of a number of tracers that have been used in patients, there is still a growing need in oncology for more specific PET radiotracers.With the development of molecular targeted therapy, several TK inhibitors have synthesized. In the seam time, the research of TK inhibitors as a precurosor of PET tracer is increasing. In recent ten years, several reversible and irreversible inhibitors, such as ML01, ML03 were labeled with fluorine-18 and carbon-11, repectively, and their potential as PET biomarkers was investigated both in vitro and in vivo. ¹⁸F-ML01, ¹¹C-ML03 has rapid in vivo metabolism, low bioavailability and, consequently, low accumulation of the labeled compound in the tumor, resulting in low tumor/blood uptake ratios. They can not used as a PET tracer for mornitoring EGFR.PD153035 (4-N-(3-bromoanilino)-6, 7-dimethoxyquinazoline, AG1517), a quinazoline derivative, has been identified as a drug for the treatment of proliferative disease. PD153035 has been shown to potently and selectively inhibit EGFR kinase activity by binding reversibly to the inner membrane ATP binding domain on the EGFR. PD153035 was proposed to be used as a PET marker in 1999 by Fredriksson et al. Although the study has provided that ¹¹C-PD153035 could accumulate in tumors, but whether the uptake signal of the "C-PD153035 correlates with the level of EGFR expression and its specific interaction has not yet been investigated. Chapter One:The uptake of PET tracer ¹¹C-PD153035 in tumors correlateswith EGFR expression levelsMethods1.Immunofluorescence StainingImmunofluorescence staining was used to show the different intensity ofEGFR in the three kinds of tumor cells lines.2. Flow Cytometry AnalysisFlow cytometry analysis was used to quantitatively detect the EGFR expression levels in the three kinds of tumor cells lines.3. Animal ModelEighty-one twelve-week-old female BalbC nude mice obtained from Animal Laboratory of the Chinese Academy (Shanghai, China) were used for the experiments. Solitary tumors were generated by inoculating 5×10⁶ viable tumor cells subcutaneously into nude mice on the lateral side of right front leg. Experiments were performed when tumors had reached the size of 10-15 mm in diameter. All studies were carried out in compliance with the national law on animal experiments and were consistent with the Guide for the Care and Use of Laboratory Animals.4.Synthesis of ¹¹C-PD153035The general procedure was performed in Tracerlab FXc system purchased from GE USA. Briefly, ¹¹C-Carbon dioxide was produced with a cyclotron using 9.6 MeV protons in the ¹⁴N (p,α) ¹¹C reaction. Radiolabeling was successfully performed by alkylation of the phenol group with ¹¹C-CH3I using standard methylation conditions. Then the reaction mixture was followed by reversed phase HPLC for purification, formulation, and sterile filtration. 5. In-vitro ¹¹C-PD153035 AccumulationOne million MDA-MB-468, A549, or MDA-MB-231 cells were seeded in 6-well culture plates with 3 ml of DMEM supplemented with 10% FCS. After 24 h incubation at 37℃, 2.1±0.8 MBq ¹¹C-PD153035 was added to the medium. After 60 min of tracer incubation, the culture medium was removed and the monolayer cells were washed 3 times with 2 mL of cold PBS. The cells were harvested from the culture plates by treatment with 0.1 ml of 0.5% trypsin for 5 min. The cells were then resuspended in 2 mL of culture medium to neutralize the trypsin. A 50 ML sample was taken to assess cell viability with trypan blue and count the number of viable cells under a microscope. The radioactivity in the cell suspensions was measured in a gamma counter and normalized to the number of viable cells in the cell suspensions. The ¹¹C-PD153035 accumulation was expressed as the percentage of the tracer dose that had accumulated per million cells.6. BiodistributionTwenty-one mice in each kind of tumor group were injected with 2. 6±0. 7 MBq of ¹¹C-PD153035 by tail vein. Five mice from each kind of tumor group were sacrifice by cervical dislocation at 10 min, 30 min and 60 min respectively. For blocking group, PD153035 (80mg/Kg) was applied (i.p) 10 min before the ¹¹C-PD153035 tail vein injection. Tumors and normal tissues were harvested, weighed, and counted in a well scintillation counter. The scintillation well counter was calibrated for these measurements with known standard activities of "C, and the activities were corrected for radioactivity decay to the time of tracer injection. The mean percentage injected per gram (%ID/g) of each samples were determined.7.In vivo PET/CT StudiesSix mice in each kind of tumor group were anesthetized by sodium pentobarbital (75 mg/Kg, i.p.) and injected with 2. 6±0. 7 MBq of ¹¹C-PD153035 by tail vein. After tail vein radiotracer injections, mice were immediately imaged in the Discovery LS PET/CT, with their long axisparallel to the transaxial plane of the tomography.8.Immunohistochemical AssaySections of three kinds of tumors were stained with haematoxylineosin (H& E) and mouse monoclonal for EGFR respectively. As a negative control,the primary antibody was omitted and replaced with PBS. Nude mice skintissue was used as a positive control for EGFR expression. Specimens wereexamined by light microscopy. All slides were assessed for EGFR expressionby two trained histopathologists who were blinded to the experimentaldesign.9. Statistical AnalysisData analyses were performed with a statistical software package SPSS 11. 5.One-way ANOVA was used for analyzing radioactivity uptake in blockingstudies. Linear Regression was performed to analyze the relation between¹¹C-PD153035 uptake and EGFR expression levels. A P value < 0.05 wasconsidered significant.Results1. Immunofluorescent examination displayed a different staining intensity in MDA-MB-468, A549 and MDA-MB-231 cell lines that was graded qualitatively intense, moderate and weak, respectively. Consistent with the results above, scoring of three cells by flow cytometry was high (83.55+1.36%, MDA-MB-468), middle (31.28±1.55%, A549) and low (5.47±1.01%, MDA-MB-231) respectively. The results of immunohistochemical assay were consisted with the results of flow cytometry analysis.2. The automated procedure enabled us to reliably and reproducibly obtain ¹¹C-PD153035 in high yields in a total synthesis time of about 38±2. Omin (n=7). The final products were obtained with 30-33% radio-chemical yield, 99% radio-chemical purity, 96% chemical purity. At end of synthesis specific radioactivity ranged from 23-45GBq/μmol. 3. ¹¹C-PD153035 accumulation in monolayers of MDA-MB-468, A549 and MDA-MB-231 cells was closely correlated with the EGFR expression levels of turaorcell lines (y=0.032+0.007x, r²=0.85; P<0.001)4. Ten minutes after injection, high activity was detected in the liver, intestine and kidney, while lower activity was observed in the lung, as well as the heart and muscle.5. Radioactivity of ¹¹C-PD153035 in tumors was correlated with the levels of EGFR expression of tumor cells too (r²=0. 63; P=0.003)6. After pretreatment with PD153035 for 10 min, ¹¹C-PD153035 accumulation reduced from 0.11±0.01 7%ID/g to 0.035±0.005 ID/g (MDA-MB-468 tumors) (F=41.76, P=0.003), 0.06±0.013%ID/cc to 0.035±0.01 ID/g (A549 tumors) (F=8.21, P=0.046) and 0.05±0.005%ID/cc to 0. 031±0. 023ID/g (MDA-MB-231 tumors) (F=6. 56, P=0.063) at 60 min p. i. ¹¹C- PD153035 respectively. There was high specific interaction between the tracer and EGFR in the three kinds of tumors.7. After finishing PET scans, the ¹¹C-PD153035 tumor uptake measured with T/NT ratio was also moderately correlated with the tracer accumulation observed in monolayer with the same tumor cells too (r²=0.48, P=0.019).Conclusions1. The automated procedure enabled us to reliably and reproducibly obtain "C-PD153035 in high yields in a total synthesis time of about 38 + 2. 0 min. The final products were obtained with 30-33% radio-chemical yield, 99% radio-chemical purity, 96% chemical purity. At end of synthesis specific radioactivity ranged from 23-45 GBq/μmol.2. Ten minutes after injection, high activity was detected in liver, intestine and kidney, while lower activity was observed in lung, as well as in heart and muscle.3. ¹¹C-PD153035 accumulation in monolayers of MDA-MB-468, A549 and MDA-MB-231 cells was closely correlated with the EGFR expression levels of tumor cell lines.4. Radioactivity of ¹¹C- PD153035 in tumors was correlated with the levels of EGFR expression in tumor cells.5. There was high specific interaction between the tracer and EGFR in the three kinds of tumors.6. ¹¹C-PD153035 has the potential as a PET tracer to yield useful information for both diagnosis and therapy of tumor. Chapter Two:Study on the Imaging of EGFR in Tumor-bearing Rat with¹¹C-PD153035 PET/CTMethods1. C6 Cell lineC6 cell line was preserved in Basic Research Center of Shandong Tumor Hospital & Institute. The cells were grown and routinely maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 units/ml penicillin, and 100μg/mL streptomycin. Cells were incubated at 37*C with 5% C02 and 95% air.2. Immunofluorescence StainingThe intensity of EGFR expression in C6 cells lines was showed by immunofluorescence staining.2. Animal ModelFive Wister rats obtained from Animal Laboratory of the Shandong University (Jinan, China) were used for the experiments. Solitary tumors were generated by inoculating 1X107 C6 cells subcutaneously into nude mice on the lateral side of both hind legs.3. Synthesis of ¹¹C-PD153035The general procedure was performed in Tracerlab FXc system purchased from GE USA.4. In-vitro blocking studyOne million C6 cells were seeded in 6-well culture plates with 3 ml of DMEM supplemented with 10% FCS. After 24 h incubation at 37℃, PD153035 was added to the medium with different concentrations (0μm, 1μm, 10μm, 100μm). After 2h of incubation, 2.1±0.8 MBq ¹¹C-PD153035 was added to the medium. After 20 min of tracer incubation, the culture medium was removed and the monolayer cells were washed 3 times with 2 mL of cold PBS. The cells were harvested from the culture plates by treatment with 0.1 ml of 0.5% trypsin for 5 min. The cells were then resuspended in 2 mL of culture medium to neutralize the trypsin. The radioactivity in the cell suspensions was measured in a gamma counter and normalized to the number of viable cells in the cell suspensions. The ¹¹C-PD153035 accumulation was expressed as the percentage of the tracer dose that had accumulated per million cells.5. PET/CT StudiesFive rats were anesthetized by sodium pentobarbital (75 mg/Kg, i. p.) and injected with 7-10 MBq of ¹¹C-PD153035 by tail vein. After radiotracer injections, rats were immediately imaged in the Discovery LS PET/CT, with their long axis parallel to the transaxial plane of the tomography. Dynamic emission scans were obtained starting at the time of injection, and continuing for 60 minutes using the following frames: 5 consecutive frames of 2min, 5 of 10min.6. PET data analysisThe kinetic of ¹¹C-PD153035 in tumor bearing rats was evaluated by region-of-interest (ROI) analysis of the serial PET images. Using Xeleris functional imaging workstation, ROIs of a standard size (25 pixels) were drown over tumors and contralateral normal muscle tissues in the appropriate transverse slices with the help of the anatomy information on CT. Radioactivity ratios (tumor/normal muscle tissue, T/NT) were calculated.7. Histopathology analysisAs soon as finished PET/CT scan, the rats were sacrificed and the tumors were excised, preserved in 10% formalin. Sections of tumors were stained with haematoxylineosin (H & E).8. Statistical AnalysisData analyses were performed with a statistical software package SPSS 11. 5. One-way ANOVA was used for analyzing radioactivity uptake in blocking studies. A P value < 0.05 was considered significant. Results1. Immunofluorescent examination displayed that thera was green staining on memberane of C6 cell lines.2. In-vitro blocking studies showed that "C-PD153035 was quickly uptaken by C6 cells (0. 052+0. 021%ID/106) and the radioactivity could be blocked by PD153035. When C6 cells were administered with the range of concentration 1μm, 10μm, 100μm of PD153035, the radioactivity uptake decreased from 0.052±0.021%ID/10⁶ to 0.045±0.006%ID/10⁶, 0.029±0.008%ID/10⁶, 0.018±0. 010%ID/10⁶ respectively. Especially with 100μm PD153035, the uptake of "C PD153035 was decreased significantly (F=6.486, P=0.003).3. The time-activity curve showed increase rapidly in first 10 min then decreased in a biexponential manner. After 60 min, the uptake appeared a tendency to leveling off. At late times the uptake of radioactivity in intestines, liver and bladder is very large and the tumors are hardly discernible.4. T/NT ratio showed an initial rapid increase in less than 10 min, followed by a continuous slow increase, peak levels (4.15) was reached within 40-50 min then a tendency to leveling off.Conclusions1. ¹¹C-PD153035 has specific interaction with EGFR in vitro.2. The time-activity curve showed increase rapidly in first 10 min then decreased in a biexponential manner. After 60 min, the uptake appeared a tendency to leveling off. The order of radioactivity in organs from high to low is liver, intestinal, kidney heart lungs and brain.3. T/NT ratio showed an initial rapid increase in less than 10 min, followed by a continuous slow increase, peak levels (4. 15) was reached within 40-50 min then a tendency to leveling off.4. ¹¹C-PD153035 has the potential to be used as a PET tumor-imaging agent for lung cancer.